This project is directed toward identifying the actual products of chemical reaction between alkylating agents which are mutagens and/or carcinogens and informational macromolecules; then assessing the role of various modifications in producing biological effects. Specifically, we plan to study the chemistry of ethylating agents, particularly the carcinogenic nitroso compounds acting on polynucleotides, RNA and DNA in vitro and on RNA and DNA derived from animal cells treated in vivo. We know from our recent work that ethylnitrosourea and ethylnitrosoguanidine primarily alkylate the oxygens, including phosphodiesters, base oxygens of C, U and T; and in the case of RNA, ribose is alkylated. Substitution of an oxygen may influence both the physical and biological properties of a nucleic acid molecule and we plan to study both facets using several experimental approaches. We have developed methods for quantitating all known alkylation products (N-substitution and all types of O-substitution) and will analyze nucleic acids, particularly from ethylnitrosourea-treated fetal rat brain cells (target cells for neoplasms during the perinatal period in rats) as compared to rat liver cells (non-target cell) under a variety of conditions since several of the alkylation reactions are very pH- and conformation-dependent and some of the products are excised at differing rates in cells. Our objective is to find whether there is a correlation between the presence or persistance of an alkyl product and carcinogenesis. We will also attempt to prepare homopolymers of O2-alkyluridine and O4-alkyluridine and O2-alkylcytidine in order to study their secondary structures and base pairing properties as one means of ascertaining whether O-alkylation of pyrimidine bases leads to abnormal base pairing in transcription. The effect of O-alkyl substitution in translation will be studied by examining the ability of modified codons to function in the binding of tRNA in the presence of ribosomes, or the ability of modified mRNA (brome mosaic virus RNA 4, coding only for the virus coat protein) to be translated accurately in a wheat embryo cell-free system.